The aim of the proposed research is to gain a better understanding of the regulatory mechanisms in smooth and skeletal muscle. In both types of muscle the contraction relaxation cycle is controlled by the intracellular concentration of Ca2 ion. Many aspects of the regulatory mechanism in skeletal muscle are known and a plausible model has been developed. The cricital features of this model are hinged on the positioning and interactions of the thin filament proteins, troponin, tropomyosin and actin. We will use fluorescence techniques to monitor these interactions, in particular the interaction of TpI with tropomyosin position will be determined by the Ca2 ion dependent interactions involving TpI; if on the other hand the model is incorrect our results will expose the inconsistencies and indicate the valid mechanism. In smooth muscle there is no universal agreement on one regulatory mechanism. The most likely mechanism is based on the Ca2-ion-dependent phosphorylation of myosin by a protein kinase. In the phosphorylated state the Mg2 ion ATPase activity of myosin is activated by actin, allowing cyclic cross-bridge interactions and the subsequent shortening or tension development. When Ca2 ion is removed, a phosphatase removes the phosphate groups from the 20,000 dalton light chains of myosin with the result that actin-activation is prevented and the muscle relaxes. Our intention is to examine this sequence of reactions and establish whether or not they constitute the regulatory mechanism in smooth muscle. Each phase will be analyzed and the extent of phosphorylation and the ATPase activity correlated. It is not known if Ca 2 ions binding to myosin also plays a role in the regulatory sequence, and we will investigate this. The protein kinase appears to be composed of several subunits, and in order to understand its mechanism of action, each subunit will be purified and characterized, and in a similar manner the phosphatase must also be examined. When certain preparative techniques are established using chicken gizzard as the smooth muscle source, we will concentrate on the regulatory mechanism in vascular tissue.